PMID- 7857690
OWN - NLM
STAT- MEDLINE
DCOM- 19950317
LR  - 20191101
IS  - 1050-3862 (Print)
IS  - 1050-3862 (Linking)
VI  - 11
IP  - 4
DP  - 1994
TI  - Development of a sensitive, highly controlled assay for molecular detection of 
      the Philadelphia chromosome in patients with chronic myelogenous leukemia.
PG  - 90-4
AB  - The Philadelphia chromosome (Ph1), present in > or = 95% of chronic myelogenous 
      leukemia (CML) patients, is a well-characterized translocation that results in a 
      unique chimeric gene product (BCR/ABL) with transforming capability. Molecular 
      methods utilizing the polymerase chain reaction (PCR) to detect BCR/ABL mRNA 
      transcripts has been useful for detecting minimal residual disease (MRD) after 
      treatment, as well as for establishing the diagnosis of CML. Amplification-based 
      assays for the BCR/ABL transcript, however, have shown variable reproducibility 
      and sensitivity. This variability may be largely due to technical differences and 
      insufficient controls. In this report, we describe the development of a highly 
      controlled, reproducible, and sensitive PCR assay to detect Ph1 that is well 
      suited to clinical and research applications. A validation study of 82 samples 
      was performed consisting of 25 dilutions of K562 cells (Ph1+) into normal 
      cultured B cells, 26 pre- and post-transplant peripheral blood samples from CML 
      patients, 16 peripheral blood samples for diagnosis of CML, and 15 peripheral 
      blood samples from healthy individuals. RNA isolated from 3 to 5 million 
      leukocytes was reverse transcribed (RT) and amplified by nested primer PCR. The 
      products were characterized using agarose gel electrophoresis. Approximately 1000 
      Ph1-positive cells admixed with 10(6) normal cells were detectable after one 
      round of amplification. In 60% of assays where one Ph1-positive cell was admixed 
      with 10(6) normal cells, a BCR/ABL product was detectable after nested primer 
      PCR. Specific measures to ensure accurate results in routine testing included (a) 
      assessing RNA integrity and adequate cDNA preparation by detection of the 
      constitutively expressed ABL mRNA, (b) monitoring sensitivity with the addition 
      and detection of K562 RNA mixed with RNA from unknown samples (failure to detect 
      the "spiked" K562 RNA indicates the presence of inhibitors or ribonucleases 
      within the unknown RNA sample), (c) detection of nucleic acid contaminants by 
      using negative controls in every assay, and (d) duplicate analysis of all samples 
      and controls. Internally, this assay was 100% reproducible. Our results verify 
      that nested primer RT-PCR is a fast, sensitive alternative to cytogenetic or 
      Southern blot analysis for monitoring MRD after treatment and for diagnosis of 
      CML. In addition, the highly controlled detection scheme presented here can be 
      used as a general model for the development of other amplification-based 
      detection assays.
FAU - Hessner, M J
AU  - Hessner MJ
AD  - Blood Center of Southeastern Wisconsin, Milwaukee 53233.
FAU - Roth, M S
AU  - Roth MS
FAU - Drobyski, W R
AU  - Drobyski WR
FAU - Baxter-Lowe, L A
AU  - Baxter-Lowe LA
LA  - eng
GR  - R29DK43470/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - Genet Anal Tech Appl
JT  - Genetic analysis, techniques and applications
JID - 9004550
RN  - 0 (RNA, Messenger)
RN  - EC 2.7.10.2 (Fusion Proteins, bcr-abl)
SB  - IM
GS  - BCR/ABL
MH  - Animals
MH  - Cattle
MH  - Fusion Proteins, bcr-abl/genetics
MH  - Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics/pathology
MH  - Neoplasm, Residual/diagnosis/genetics
MH  - *Philadelphia Chromosome
MH  - Predictive Value of Tests
MH  - RNA, Messenger/genetics
MH  - Reproducibility of Results
MH  - Tumor Cells, Cultured
EDAT- 1994/01/01 00:00
MHDA- 1994/01/01 00:01
CRDT- 1994/01/01 00:00
PHST- 1994/01/01 00:00 [pubmed]
PHST- 1994/01/01 00:01 [medline]
PHST- 1994/01/01 00:00 [entrez]
AID - 1050-3862(94)90045-0 [pii]
AID - 10.1016/1050-3862(94)90045-0 [doi]
PST - ppublish
SO  - Genet Anal Tech Appl. 1994;11(4):90-4. doi: 10.1016/1050-3862(94)90045-0.